The antigen 43 structure reveals a molecular velcro-like mechanism of autotransporter-mediated bacterial clumping

Aggregation and biofilm formation are critical mechanisms for bacterial resistance to host immune factors and antibiotics. Autotransporter (AT) proteins, which represent the largest group of outer-membrane and secreted proteins in Gram-negative bacteria, contribute significantly to these phenotypes. Despite their abundance and role in bacterial pathogenesis, most AT proteins have not been structurally characterized, and there is a paucity of detailed information with regard to their mode of action. Here we report the structure–function relationships of Antigen 43 (Ag43a), a prototypic self-associating AT protein from uropathogenic Escherichia coli. The functional domain of Ag43a displays a twisted L-shaped β-helical structure firmly stabilized by a 3D hydrogen-bonded scaffold. Notably, the distinctive Ag43a L shape facilitates self-association and cell aggregation. Combining all our data, we define a molecular “Velcro-like” mechanism of AT-mediated bacterial clumping, which can be tailored to fit different bacterial lifestyles such as the formation of biofilms.

Molecular analysis of the acinetobacter baumannii biofilm-associated protein

Acinetobacter baumannii is a multidrug-resistant pathogen associated with hospital outbreaks of infection across the globe, particularly in the intensive care unit. The ability of A. baumannii to survive in the hospital environment for long periods is linked to antibiotic resistance and its capacity to form biofilms. Here we studied the prevalence, expression, and function of the A. baumannii biofilm-associated protein (Bap) in 24 carbapenem-resistant A. baumannii ST92 strains isolated from a single institution over a 10-year period. The bap gene was highly prevalent, with 22/24 strains being positive for bap by PCR. Partial sequencing of bap was performed on the index case strain MS1968 and revealed it to be a large and highly repetitive gene approximately 16 kb in size. Phylogenetic analysis employing a 1,948-amino-acid region corresponding to the C terminus of Bap showed that BapMS1968 clusters with Bap sequences from clonal complex 2 (CC2) strains ACICU, TCDC-AB0715, and 1656-2 and is distinct from Bap in CC1 strains. By using overlapping PCR, the bapMS1968 gene was cloned, and its expression in a recombinant Escherichia coli strain resulted in increased biofilm formation. A Bap-specific antibody was generated, and Western blot analysis showed that the majority of A. baumannii strains expressed an ∼200-kDa Bap protein. Further analysis of three Bap-positive A. baumannii strains demonstrated that Bap is expressed at the cell surface and is associated with biofilm formation. Finally, biofilm formation by these Bap-positive strains could be inhibited by affinity-purified Bap antibodies, demonstrating the direct contribution of Bap to biofilm growth by A. baumannii clinical isolates.

Molecular characterization of endocarditis-associated Staphylococcus aureus

Infective endocarditis (IE) is a life-threatening infection of the heart endothelium and valves. Staphylococcus aureus is a predominant cause of severe IE and is frequently associated with infections in health care settings and device-related infections. Multilocus sequence typing (MLST), spa typing, and virulence gene microarrays are frequently used to classify S. aureus clinical isolates. This study examined the utility of these typing tools to investigate S. aureus epidemiology associated with IE. Ninety-seven S. aureus isolates were collected from patients diagnosed with (i) IE, (ii) bloodstream infection related to medical devices, (iii) bloodstream infection not related to medical devices, and (iv) skin or soft-tissue infections. The MLST clonal complex (CC) for each isolate was determined and compared to the CCs of members of the S. aureus population by eBURST analysis. The spa type of all isolates was also determined. A null model was used to determine correlations of IE with CC and spa type. DNA microarray analysis was performed, and a permutational analysis of multivariate variance (PERMANOVA) and principal coordinates analysis were conducted to identify genotypic differences between IE and non-IE strains. CC12, CC20, and spa type t160 were significantly associated with IE S. aureus. A subset of virulence-associated genes and alleles, including genes encoding staphylococcal superantigen-like proteins, fibrinogen-binding protein, and a leukocidin subunit, also significantly correlated with IE isolates. MLST, spa typing, and microarray analysis are promising tools for monitoring S. aureus epidemiology associated with IE. Further research to determine a role for the S. aureus IE-associated virulence genes identified in this study is warranted.

Molecular characterization of the EhaG and UpaG trimeric autotransporter proteins from pathogenic Escherichia coli

Trimeric autotransporter proteins (TAAs) are important virulence factors of many Gram-negative bacterial pathogens. A common feature of most TAAs is the ability to mediate adherence to eukaryotic cells or extracellular matrix (ECM) proteins via a cell surface-exposed passenger domain. Here we describe the characterization of EhaG, a TAA identified from enterohemorrhagic Escherichia coli (EHEC) O157:H7. EhaG is a positional orthologue of the recently characterized UpaG TAA from uropathogenic E. coli (UPEC). Similarly to UpaG, EhaG localized at the bacterial cell surface and promoted cell aggregation, biofilm formation, and adherence to a range of ECM proteins. However, the two orthologues display differential cellular binding: EhaG mediates specific adhesion to colorectal epithelial cells while UpaG promotes specific binding to bladder epithelial cells. The EhaG and UpaG TAAs contain extensive sequence divergence in their respective passenger domains that could account for these differences. Indeed, sequence analyses of UpaG and EhaG homologues from several E. coli genomes revealed grouping of the proteins in clades almost exclusively represented by distinct E. coli pathotypes. The expression of EhaG (in EHEC) and UpaG (in UPEC) was also investigated and shown to be significantly enhanced in an hns isogenic mutant, suggesting that H-NS acts as a negative regulator of both TAAs. Thus, while the EhaG and UpaG TAAs contain some conserved binding and regulatory features, they also possess important differences that correlate with the distinct pathogenic lifestyles of EHEC and UPEC.

Molecular characterization of Escherichia coli strains that cause symptomatic and asymptomatic urinary tract infections

The differences between Escherichia coli strains associated with symptomatic and asymptomatic urinary tract infections (UTIs) remain to be properly determined. Here we examined the prevalence of plasmid types and bacteriocins, as well as genetic relatedness, in a defined collection of E. coli strains that cause UTIs. Comparative analysis identified a subgroup of strains with a high number of virulence genes (VGs) and microcins M/H47. We also identified associations between microcin genes, VGs, and specific plasmid types.

Molecular characterization of UpaB and UpaC, two new autotransporter proteins of uropathogenic Escherichia coli CFT073

Uropathogenic Escherichia coli (UPEC) is the primary cause of urinary tract infection (UTI) in the developed world. The major factors associated with virulence of UPEC are fimbrial adhesins, which mediate specific attachment to host receptors and trigger innate host responses. Another group of adhesins is represented by the autotransporter (AT) subgroup of proteins. The genome-sequenced prototype UPEC strain CFT073 contains 11 putative AT-encoding genes. In this study, we have performed a detailed molecular characterization of two closely related AT adhesins from CFT073: UpaB (c0426) and UpaC (c0478). PCR screening revealed that the upaB and upaC AT-encoding genes are common in E. coli. The upaB and upaC genes were cloned and characterized in a recombinant E. coli K-12 strain background. This revealed that they encode proteins located at the cell surface but possess different functional properties: UpaB mediates adherence to several ECM proteins, while UpaC expression is associated with increased biofilm formation. In CFT073, upaB is expressed while upaC is transcriptionally repressed by the global regulator H-NS. In competitive colonization experiments employing the mouse UTI model, CFT073 significantly outcompeted its upaB (but not upaC) isogenic mutant strain in the bladder. This attenuated phenotype was also observed in single-challenge experiments, where deletion of the upaB gene in CFT073 significantly reduced early colonization of the bladder.

Molecular analysis of type 3 fimbrial genes from Escherichia coli, Klebsiella and Citrobacter species

Background Catheter-associated urinary tract infection (CAUTI) is the most common nosocomial infection in the United States and is caused by a range of uropathogens. Biofilm formation by uropathogens that cause CAUTI is often mediated by cell surface structures such as fimbriae. In this study, we characterised the genes encoding type 3 fimbriae from CAUTI strains of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Citrobacter koseri and Citrobacter freundii. Results Phylogenetic analysis of the type 3 fimbrial genes (mrkABCD) from 39 strains revealed they clustered into five distinct clades (A-E) ranging from one to twenty-three members. The majority of sequences grouped in clade A, which was represented by the mrk gene cluster from the genome sequenced K. pneumoniae MGH78578. The E. coli and K. pneumoniae mrkABCD gene sequences clustered together in two distinct clades, supporting previous evidence for the occurrence of inter-genera lateral gene transfer. All of the strains examined caused type 3 fimbriae mediated agglutination of tannic acid treated human erythrocytes despite sequence variation in the mrkD-encoding adhesin gene. Type 3 fimbriae deletion mutants were constructed in 13 representative strains and were used to demonstrate a direct role for type 3 fimbriae in biofilm formation. Conclusions The expression of functional type 3 fimbriae is common to many Gram-negative pathogens that cause CAUTI and is strongly associated with biofilm growth. Our data provides additional evidence for the spread of type 3 fimbrial genes by lateral gene transfer. Further work is now required to substantiate the clade structure reported here by examining more strains as well as other bacterial genera that make type 3 fimbriae and cause CAUTI.

European aspects of standard setting in occupational hygiene and medicine

Occupational standards concerning the allowable concentrations of chemical compounds in the ambient air of workplaces have been established in several countries at national levels. With the integration of the European Union, a need exists for establishing harmonized Occupational Exposure Limits. For analytical developments, it is apparent that methods for speciation or fractionation of carcinogenic metal compounds will be of increasing practical importance for standard setting. Criteria of applicability under field conditions, cost-effectiveness, and robustness are practical driving forces for new developments. When the European Union issued a list of 62 chemical substances with Occupational Exposure Limits in 2000, 25 substances received a 'skin' notation. The latter indicates that toxicologically significant amounts may be taken up via the skin. Similar notations exist on national levels. For such substances, monitoring concentrations in ambient air will not be sufficient; biological monitoring strategies will gain further importance in the medical surveillance of workers who are exposed to such compounds. Proceedings in establishing legal frameworks for a biological monitoring of chemical exposures within Europe are paralleled by scientific advances in this field. A new aspect is the possibility of a differential adduct monitoring, using blood proteins of different half-life or lifespan. This technique allows differentiation between long-term mean exposure to reactive chemicals and short-term episodes, for example, by accidental overexposure. For further analytical developments, the following issues have been addressed as being particularly important: New dose monitoring strategies, sensitive and reliable methods for detection of DNA adducts, cytogenetic parameters in biological monitoring, methods to monitor exposure to sensitizing chemicals, and parameters for individual susceptibilities to chemical toxicants.

Effects of HNO3 molecular doping in graphene/Si Schottky barrier solar cells

Schottky barrier solar cells based on graphene/n-silicon heterojunction have been fabricated and characterized and the effect of graphene molecular doping by HNO3 on the solar cells performances have been analyzed. Different doping conditions and thermal annealing processes have been tested to asses and optimize the stability of the devices. The PCE of the cells increases after the treatment by HNO3 and reaches 5% in devices treated at 200 °C immediately before the exposition to the oxidant. Up to now our devices retain about 80% of efficiency over a period of two weeks, which represents a good stability result for similar devices.

Prescribing for nausea in palliative care : A cross-sectional national survey of Australian palliative medicine doctors

Background: Nausea can be a debilitating symptom for patients with a life-limiting illness. While addressing reversible components, nonpharmacological strategies and antiemetics are the main therapeutic option. The choice of medication, dose, and route of administration remain highly variable. Objective: The aim of this study was to codify the current clinical approaches and quantify any variation found nationally. Methods: A cross-sectional study utilizing a survey of palliative medicine clinicians examined prescribing preferences for nausea using a clinical vignette. Respondent characteristics, the use of nonpharmacological interventions, first- and second-line antiemetic choices, commencing and maximal dose, and time to review were collected. Results: Responding clinicians were predominantly working in palliative medicine across a range of settings with a 49% response rate (105/213). The main nonpharmacological recommendation was “small, frequent snacks.” Metoclopramide was the predominant first-line agent (69%), followed by haloperidol (26%), while second-line haloperidol was the predominant agent (47%), with wide variation in other nominated agents. Respondents favoring metoclopramide as first-line tended to use haloperidol second-line (65%), but not vice versa. Maximal doses for an individual antiemetic varied up to tenfold. Conclusion: For nausea, a commonly encountered symptom in palliative care, clinicians' favored metoclopramide and haloperidol; however, after these choices, there was large variation in antiemetic selection. While most clinicians recommended modifying meal size and frequency, use of other nonpharmacological therapies was limited.

The adhesion molecule L1 regulates transendothelial migration and trafficking of dendritic cells

The adhesion molecule L1, which is extensively characterized in the nervous system, is also expressed in dendritic cells (DCs), but its function there has remained elusive. To address this issue, we ablated L1 expression in DCs of conditional knockout mice. L1-deficient DCs were impaired in adhesion to and transmigration through monolayers of either lymphatic or blood vessel endothelial cells, implicating L1 in transendothelial migration of DCs. In agreement with these findings, L1 was expressed in cutaneous DCs that migrated to draining lymph nodes, and its ablation reduced DC trafficking in vivo. Within the skin, L1 was found in Langerhans cells but not in dermal DCs, and L1 deficiency impaired Langerhans cell migration. Under inflammatory conditions, L1 also became expressed in vascular endothelium and enhanced transmigration of DCs, likely through L1 homophilic interactions. Our results implicate L1 in the regulation of DC trafficking and shed light on novel mechanisms underlying transendothelial migration of DCs. These observations might offer novel therapeutic perspectives for the treatment of certain immunological disorders.

Vibrational spectroscopy of the borate mineral priceite : implications for the molecular structure

Priceite is a calcium borate mineral and occurs as white crystals in the monoclinic pyramidal crystal system. We have used a combination of Raman spectroscopy with complimentary infrared spectroscopy and scanning electron microscopy with Energy-dispersive X-ray Spectroscopy (EDS) to study the mineral priceite. Chemical analysis shows a pure phase consisting of B and Ca only. Raman bands at 956, 974, 991, and 1019 cm−1 are assigned to the BO stretching vibration of the B10O19 units. Raman bands at 1071, 1100, 1127, 1169, and 1211 cm−1 are attributed to the BOH in-plane bending modes. The intense infrared band at 805 cm−1 is assigned to the trigonal borate stretching modes. The Raman band at 674 cm−1 together with bands at 689, 697, 736, and 602 cm−1 are assigned to the trigonal and tetrahedral borate bending modes. Raman spectroscopy in the hydroxyl stretching region shows a series of bands with intense Raman band at 3555 cm−1 with a distinct shoulder at 3568 cm−1. Other bands in this spectral region are found at 3221, 3385, 3404, 3496, and 3510 cm−1. All of these bands are assigned to water stretching vibrations. The observation of multiple bands supports the concept of water being in different molecular environments in the structure of priceite. The molecular structure of a natural priceite has been assessed using vibrational spectroscopy.

Circulating fragments of N-Terminal Pro-B-Type Natriuretic Peptides in plasma of heart failure patients

BACKGROUND: The use of nonstandardized N-terminal pro-B-type natriuretic peptide (NT-proBNP) assays can contribute to the misdiagnosis of heart failure (HF). Moreover, there is yet to be established a common consensus regarding the circulating forms of NT-proBNP being used in current assays. We aimed to characterize and quantify the various forms of NT-proBNP in the circulation of HF patients. METHODS: Plasma samples were collected from HF patients (n = 20) at rest and stored at -80 degrees C. NT-proBNP was enriched from HF patient plasma by use of immunoprecipitation followed by mass spectrometric analysis. Customized homogeneous sandwich AlphaLISA (R) immunoassays were developed and validated to quantify 6 fragments of NT-proBNP. RESULTS: Mass spectrometry identified the presence of several N- and C-terminally processed forms of circulating NT-proBNP, with physiological proteolysis between Pro2-Leu3, Leu3-Gly4, Pro6-Gly7, and Pro75-Arg76. Consistent with this result, AlphaLISA immunoassays demonstrated that antibodies targeting the extreme N or C termini measured a low apparent concentration of circulating NT-proBNP. The apparent circulating NT-proBNP concentration was increased with antibodies targeting nonglycosylated and nonterminal epitopes (P < 0.05). CONCLUSIONS: In plasma collected from HF patients, immunoreactive NT-proBNP was present as multiple N- and C-terminally truncated fragments of the full length NT-proBNP molecule. Immunodetection of NT-proBNP was significantly improved with the use of antibodies that did not target these terminal regions. These findings support the development of a next generation NT-proBNP assay targeting nonterminal epitopes as well as avoiding the central glycosylated region of this molecule. (c) 2013 American Association for Clinical Chemistry